Glycosylation status of vitamin D binding
protein in cancer patients
Douglas S. Rehder, Randall W. Nelson, and Chad R. Borges*
Molecular Biomarkers, The Biodesign Institute at Arizona State University, Tempe, Arizona 85287
Received 19 March 2009; Revised 30 June 2009; Accepted 14 July 2009
DOI: 10.1002/pro.214
Published online 29 July 2009 proteinscience.org
Abstract: On the basis of the results of activity studies, previous reports have suggested that
vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer
patients, eliminating the molecular precursor of the immunologically important Gc macrophage
activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation
was to directly determine the relative degree of O-linked trisaccharide glycosylation of serumderived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results
obtained by electrospray ionization-based mass spectrometric immunoassay showed that there
was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined
relative to healthy controls. These results suggest that alternative hypotheses regarding the
molecular and/or structural origins of GcMAF must be considered to explain the relative inability of
cancer patient serum to activate macrophages.
Keywords: vitamin D binding protein; GcMAF; cancer; glycosylation
Introduction
Gc macrophage activating factor (GcMAF) is a naturally derived form of human vitamin D binding protein
that has recently shown notable clinical value as a
potent immunotherapy against human breast, prostate,
and colorectal cancer.1–3 Molecularly, GcMAF is
thought to be DBP with a single terminal O-linked
GalNAc sugar residue.4–7 In its native molecular state,
DBP is a mixture of posttranslationally unmodified
(except for disulfide bonds) and O-glycosylated species, the glycosidic degree and nature of which
depends on DBP genotype.8–13 For humans, there are
Abbreviations: CDI, 1,10 -carbonyldiimidazole; DBP, vitamin D
binding protein; ESI-MSIA, electrospray ionization-based mass
spectrometric immunoassay; Gal, galactose; GalNAc, Nacetylgalactosamine; GcMAF, Gc macrophage activating factor;
HBS,
hepes-buffered
saline;
MES,
2-(N-morpholino)
ethanesulfonic acid; nagalase, a-N-acetylgalactosaminidase;
NeuNAc, N-acetylneuraminic acid.
Agilent Technologies Foundation; Research Project Gift 08US422UR.
*Correspondence to: Chad R. Borges, Molecular Biomarkers,
The Biodesign Institute at Arizona State University, P.O. Box
876601, Tempe, AZ 85287. E-mail: [email protected]
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PROTEIN SCIENCE 2009 VOL 18:2036—2042
three major allelic variants designated Gc1F, Gc1S, and
Gc2. Relative to the Gc1F protein, a D416E mutation
defines the Gc1S protein, and a T420K mutation
defines the Gc2 protein. The Gc2 mutation removes
the major site for O-linked trisaccharide glycosylation,10,14 a fact which has recently been verified by
mass spectrometric analysis of the intact protein.8,9 All
major allelic forms of DBP can be converted to GcMAF
(by the action of b-galactosidase of inflammation-activated B cells and, for Gc1 proteins, inflammation-activated T-cell sialidase4–7) despite the fact that Gc2 lacks
O-linked trisaccharidic glycosylation,4,5 suggesting the
presence of a low occupation, alternative glycosylation
site on Gc2 proteins (which must be genetically conserved in Gc1F and Gc1S proteins).4,5,8
Studies in cancer patients have shown that highserum levels of a-N-acetylgalactosaminidase (nagalase)
activity correspond in linear fashion to both tumor
burden15,16 (in mice and humans) and, inversely,
GcMAF ‘‘precursor activity’’ in humans.2,3 This ‘‘precursor activity’’ has been assigned (without direct
structural evidence) as the quantity of O-linked trisaccharide glycosylated DBP available in patient plasma,
and is reported to be significantly depleted or even
eliminated in cancer patients based on activity studies.1–3,15,17 Since direct structural evidence for this
C 2009 The Protein Society
Published by Wiley-Blackwell. V
assignment has not previously been obtained, the purpose of this investigation was to assess the relative
degree of O-linked trisaccharide glycosylation of DBP
in breast, colorectal, pancreatic, and prostate cancer
patients compared with healthy individuals.
Results
Charge state deconvoluted ESI mass spectra of immunoaffinity-isolated, intact DBP from the serum or
plasma of 56 cancer patients (nine breast cancer, 11
colorectal cancer, nine prostate cancer, and 27 pancreatic cancer patients) and 29 healthy patients (exemplified in Fig. 1) demonstrate no significant differences
with regard to the relative degree of O-linked trisaccharide glycosylation compared with healthy individuals (see Fig. 2). The data from the healthy individuals
are in agreement with data published recently on the
relative degree of DBP trisaccharide glycosylation
assessed in the plasma of over 100 noncancerous
individuals.8
In 1983, Viau et al.13 characterized the O-glycan
trisaccharide of DBP as NeuNAc a-(2!3) Gal b-(1!3)
GalNAc a-(1!0). Thus, mass shifts of þ162 and þ203
Da described later in reference to enzymatic glycosylation are assumed to represent Gal and GalNAc, respectively; even though mass shifts alone cannot ab initio
distinguish between galactose and mannose or N-acetylgalactosamine and N-acetylglucosamine.
Mass spectra shown in Figure 1 also provide initial evidence, by virtue of a precise þ203 Da mass
shift (corresponding to the presence of a terminal GalNAc residue8,13), that significant quantities of what
might nominally be considered GcMAF exist in the serum of 15 of 29 cancer patients. On the basis of the
relative peak areas and the known concentration range
of DBP in plasma, the approximate concentration of
this molecular species in cancer patients is 5–10 mg/
L (100–200 nM). As compared in Figure 3, the spectra from 21 of 29 age and gender matched healthy
patients also exhibited peaks indicating the presence
of a single residual GalNAc residue attached to Gc1
DBP. When observed, the relative quantity of Gc1 DBP
carrying a single GalNAc residue was found to be
greater in cancer patients than healthy individuals
(P < 0.02; Fig. 3). Out of 32 observations of Gc2 allele
products, no peaks were observed that could possibly
represent Gc2 protein molecules carrying this single
residual GalNAc residue, even when found paired in
heterozygous fashion with Gc1 protein carrying the residual GalNAc residue.
Discussion
Trisaccharide glycosylated-DBP is not
eliminated in cancer patients
The results presented here are in apparent conflict
with the proposed mechanism for the inability of cancer patient serum to produce GcMAF on exposure to
Rehder et al.
b-galactosidase and sialidase1–3,15,17: According to the
theory, trisaccharide glycosylated-DBP in cancer
patients is depleted to the point where it should be
undetectable by the method employed in these studies.1–3,15,17 The depletion effect should be dramatic
because a small mass spectral peak representing, for
example, a 1% relative abundance of trisaccharide glycosylated-DBP would still correspond to relatively
large quantities of GcMAF precursor (trisaccharide glycosylated-DBP) in the patient serum, on the order of
4 mg/L (based on a total serum concentration of
400 mg/L14), a quantity much greater than the 100
ng per person quantities of GcMAF employed as an
effective cancer therapeutic.1–3 Thus, the observation
that there is no depletion of trisaccharide glycosylatedDBP in cancer patients means that trisaccharide glycosylated-DBP cannot be the direct precursor of GcMAF
that is eliminated in cancer patients.
Redefining the molecular definition
and origin of GcMAF
The presence of relatively abundant quantities (5–10
mg/L or 100–200 nM) of what might nominally be
considered GcMAF (i.e., DBP modified by a single terminal GalNAc residue) in cancer patient serum at first
seems to both contradict macrophage activation studies using cancer patient serum1–3,15,17 and present a
paradox in which cancer patients with relatively high
circulating concentrations of GcMAF can effectively be
treated with trace quantities of GcMAF.1–3
These apparent contradictions may be avoided by
proposing that the most likely explanation for the
apparent observation of GcMAF in the serum of Gc1
allele-carrying cancer patients is that the observed
DBP molecules with a single terminal GalNAc at T420
are not GcMAF, that is, are not the same molecular
species responsible for the macrophage activation
observed by Yamamoto et al.1–3,15,17 This could be the
case if, for example, genuine GcMAF carries a T418linked GalNAc monosaccharide (derived from Gal-GalNAc-T418-modified DBP, as it most likely is for Gc2
allele products4,5), and the GalNAc-modified DBP species directly observed in these studies were derived
from T420-linked trisaccharide glycosylated-DBP. The
T420 linkage site is suspected for the directly observed
GalNAc-modified DBP because the genetic variation of
human DBP creates a natural site-directed mutagenesis study in which T420 is eliminated to create the
Gc2 allele product. In the 32 cases of Gc2 allele product examined in this study, none were found to carry
the single GalNAc residue observed on many Gc1 allele
products-even when Gc2 protein was found paired in
heterozygous fashion with Gc1 protein carrying the residual GalNAc residue. (Notably, no Gc2 allele products were found to carry the trisaccharide either.)
These data suggest that the GalNAc monosaccharide
observed is attached to T420-the same site of
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Figure 1. Charge state deconvoluted ESI mass spectra of intact DBP isolated directly from the blood plasma of 3 cancer
patients and 3 healthy individuals with matching DBP diploid genotype. The base peaks at approximately 51,200 Da
represent unmodified DBP (which varies in exact mass according to DBP genotype), a peak at Dm þ 162 Da, if present,
represents nonenzymatically glycated DBP, a peak at Dm þ 203.2, if present, represents DBP modified by a single GalNAc
residue, a broadened peak at approximately Dm þ 365.4 likely represents an O-linked disaccharide (DS) glycoform
(Gal1GalNAc1),8 and a peak at Dm þ 656.6 represents the O-linked trisaccharide glycoform (TS, NeuNAc1Gal1GalNAc1).8,13
Displayed masses correspond to the observed m/z value of an ‘‘MHþ’’ species. (A) Homozygous 1F/1F DBP from a breast
cancer patient (calculated unmodified 1F DBP m/z ¼ 51,189.2) (B) Homozygous 1F/1F DBP from a healthy individual (C)
Homozygous 1S/1S DBP from a colorectal cancer patient (calculated unmodified 1S DBP m/z ¼ 51203.2) (D) Homozygous
1S/1S DBP from a healthy individual (E) Homozygous Gc2/Gc2 DBP from a colorectal cancer patient (calculated unmodified
Gc2 DBP m/z ¼ 51216.3) (F) Homozygous Gc2/Gc2 DBP from a healthy individual. Though not shown directly, the mass
resolution is sufficient to readily classify the diploid genotype of all heterozygous combinations of DBP allele products.
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Glycosylation of DBP in Cancer
Figure 2. Allele-specific relative abundance of DBP O-linked trisaccharide glycosylation in cancer patients compared to
healthy individuals. n-values for each sub-group are as follows: Total Healthy Individuals ¼ 29, Total Cancer Patients ¼ 56;
1F/1F Healthy ¼ 3, 1F/1F Cancer ¼ 4; 1S/1S Healthy ¼ 10, 1S/1S Cancer ¼ 15, Gc2/Gc2 Healthy ¼ 4, Gc2/Gc2 Cancer ¼ 4;
1F/1S Healthy ¼ 3, 1F/1S Cancer ¼ 10, 1S/Gc2 Healthy ¼ 5, 1S/Gc2 Cancer ¼ 18; 1F/Gc2 Healthy ¼ 3, Cancer ¼ 3. A few
rare, unclassified DBP genotypic variants were also observed. Error bars indicate sample standard deviation. There are no
statistically significant differences between cancer patients and healthy controls for any diploid genotype. As depicted in the
plot and shown in raw format in Figure 1, Gc2 allele products completely lack trisaccharide glycosylation; the presence of
trisaccharide glycosylated-DBP in heterozygous individuals is unambiguously due to modification of 1F or 1S DBP.8,9
NeuNAc-Gal-GalNAc-Thr glycosylation suggested in
previous studies by other investigators.13,14,18 With
regard to glycosylation at T418, previous work of ours
using tandem mass spectrometry of DBP peptides has
confirmed trisaccharide glycosylation of T420 and has
gone on to show that a disaccharide glycosylated form
of DBP appears, in some Gc1-derived protein molecules, to have the carbohydrate attached at T418.8
Thus, glycosylation of Gc1 allele products at T418 may
be involved with formation of active GcMAF and may
serve as a way out of the apparent contradictions
between the data presented here and conclusions of
molecular structure drawn from activity studies.1–3,15,17
Due to its extremely low natural abundance, it is
unlikely that true GcMAF from any allele product
would be detected by MSIA without isolation from the
most abundant forms of DBP.
Additionally, it may be that the existence of the
observed GalNAc-modified DBP (which is too abundant in cancer patients to be GcMAF) solves the contradictory reports of Kanan et al.19 and Yamamoto
et al.5–7,17,20 The former found noninducible generation of GalNAc-only modified DBP via lectin-based immunoassay which, when believed to be GcMAF, is in
conflict with the activity based studies of Yamamoto
et al. which suggest that a mediator of inflammation
Rehder et al.
Figure 3. Relative percent of DBP modified with a GalNAc
monosaccharide at T420 in cancer patients compared to
age and gender matched healthy individuals. Data were
tabulated only when a mass spectral peak representing the
target species was observed (i.e., samples with a relative
percentage of zero are not included). This molecular
species was not observed on any of the 32 observed Gc2
allele products-even when found paired in heterozygous
fashion with Gc1 protein carrying the residual GalNAc
residue-hence the T420 positional assignment. Error bars
represent standard deviation. *P < 0.02, Student’s t-test.
PROTEIN SCIENCE VOL 18:2036—2042
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such as lysophosphatidylcholine must be present to
induce production of GcMAF. The GalNAc monosaccharide-modified DBP observed in this report would
produce positive results in a lectin-based immunoassay
performed on samples tested in the absence of a mediator of inflammation, showing itself to be ‘‘noninducible.’’ Yet, as described earlier, this ‘‘noninducible’’ species is far too abundant in cancer patients to be active
GcMAF.
Methods
Electrospray ionization-based mass spectrometric immunoassay (ESI-MSIA) was employed to ascertain the
relative degree of trisaccharidic glycosylation of serum
or plasma DBP in 56 human cancer patients (nine
breast cancer, 11 colorectal cancer, nine prostate cancer, and 27 pancreatic cancer patients) and 29 healthy
humans.
MSIA is a high throughput-amenable analytical
technique in which a native protein of interest is purified by immunoprecipitation then analyzed intact by
mass spectrometry. Since its inception in 1995 by
R.W. Nelson et al.,21 it has been applied to study a variety of proteins and peptides,22–36 and has served as
the foundation for the nascent field of Population Proteomics.37–42 Roughly, MSIA can be envisioned as
ultra high resolution, semiquantitative Western blotting in which protein variants are thoroughly resolved
and their molecular masses determined to within less
than 2 Da.
Materials
Polyclonal rabbit anti-human DBP (GC-Globulin) antibodies (Cat. No. A0021) were obtained from DAKO
(Carpinteria, CA). According to the manufacturer’s
specifications, this antibody is for in vitro diagnostic
use and is intended for the determination of DBP in
gel immunoprecipitation techniques and for phenotyping of DBP by immunofixation. Premixed MES-buffered saline powder packets were from Pierce (Rockford, IL). Isolation of DBP from plasma was carried
out with proprietary MSIA pipette tips from Intrinsic
Bioprobes (Tempe, AZ) derivatized with the DBP antibodies via 1,10 -carbonyldiimidazole (CDI) chemistry as
previously described.33,43 Serum samples from 29 cancer patients (nine breast cancer, 11 colorectal cancer,
nine prostate cancer) of African-American, Caucasian,
and Hispanic origin, along with age, gender, and racematched controls were purchased from ProMedDx
(Norton, MA). Twenty seven plasma samples from
pancreatic cancer patients were generously donated by
Dr. Douglas Lake of the School of Life Sciences at Arizona State University. All cancer patients were diagnosed by qualified physicians. Protein Captrap
catridges for LC/MS were obtained from Michrom
Bioresources (Auburn, CA). Premade 10 mM Hepesbuffered saline (HBS) was bought from Biacore
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(Piscataway, NJ). All other chemicals were obtained
from Sigma-Aldrich (St. Louis, MO).
Sample preparation for the analysis of intact
DBP by MSIA ESI-TOF-MS
With the aid of a Beckman Multimek 96-channel automated pipetter, MSIA pipette tips that had been preactivated with CDI33,43 were derivatized with polyclonal
DBP antibodies by repetitively flowing (aspirating and
dispensing 750 times) 50 lL volumes of antibody solution (150 lL/well; 0.05 g/L in 0.1M MES buffered saline, pH 4.7) through the tips. Antibody-linked tips
were stored in HBS at 4 C until the day of use at
which time they were prerinsed (400 lL/well; 150 lL
aspirate and dispense cycles; 10 cycles) with HBS then
used to extract DBP from individual samples (25 lL of
human serum or plasma diluted with 75 lL of HBS) at
room temperature (100 lL/well; 50 lL aspirate and
dispense cycles; 750 cycles). Pipette tips were then
ejected from the robot and allowed to sit in their respective plasma samples at room temperature until
they were individually (manually) washed (by drawing
from a fresh reservoir of liquid and dispensing to
waste) and eluted as follows: Five cycles of 200 lL of
HBS, five cycles of 200 lL distilled water, five cycles
200 lL of 2M ammonium acetate/acetonitrile (3:1 v/v),
ten cycles of 200 lL of distilled water. Elution was
accomplished by briefly air-drying the pipette frits
then drawing 5 lL of a mixture of 100% formic acid/
acetonitrile/distilled water (9/5/1 v/v/v), mixing over
the pipette affinity capture frit for 20–30 seconds, and
dispensing into a 96-conical well polypropylene autosampler tray. Frits were then washed with an additional
5.5 lL distilled water which was used to dilute the
eluted sample. Six microliters was injected into the LCTOF-MS well within 10 min of elution to avoid protein
formylation or other potential modifications from the
elution solvent.
ESI-TOF-MS
A trap-and-elute form of sample concentration/solvent
exchange rather than traditional LC was used for these
analyses. Six-microliter samples were injected by a
Spark Holland Endurance autosampler in microliter
pick-up mode and loaded by an Eksigent nanoLC*1D
at 10 lL/min (90/10 water/acetonitrile containing
0.1% formic acid, Solvent A) onto a protein captrap
(polymeric/reversed phase sorbent, Michrom Bioresources, Auburn, CA) configured for unidirectional
flow on a 6-port divert valve. After 2 min, the divert
valve position was automatically toggled and flow rate
over the protein captrap cartridge changed to 1 lL/
min solvent A (running directly to the ESI inlet) which
was immediately ramped over 8 min of a linear gradient from 10 to 90% solvent B (100% acetonitrile). By
10.2 min, the analysis was completed and the flow rate
back to 100% solvent A.
Glycosylation of DBP in Cancer
DBP and its variants eluted between 5.5 and 7.5
min into a Bruker MicrOTOF-Q (Q-TOF) mass spectrometer operating in positive ion, TOF-only mode,
acquiring spectra in the m/z range of 50–3000. ESI
settings for the Agilent G1385A capillary nebulizer ion
source were as follows: End Plate Offset 500 V, Capillary 4500 V, Nebulizer nitrogen 2 Bar, Dry Gas
nitrogen 3.0 L/min at 225 C. Data were acquired in
profile mode at a digitizer sampling rate of 2 GHz.
Spectra rate control was by summation at 1 Hz.
Data analysis for intact DBP
Approximately 1 min of recorded spectra were averaged across the chromatographic peak apex of DBP
elution. The ESI charge-state envelope was deconvoluted with Bruker DataAnalysis v3.4 software to a
mass range of 1000 Da on either side of any identified
peak. Deconvoluted spectra were baseline subtracted
and all peaks were integrated. Tabulated mass spectral
peak areas were exported to a spreadsheet for further
calculation and determination of the peak areas of interest relative to all other forms of DBP present in the
mass spectrum.
Conclusions
Trisaccharide glycosylated-DBP circulates in abundant
quantities (comparable to that observed in healthy
individuals) in the blood of breast, colorectal, pancreatic, and prostate cancer patients, making it unlikely
that it is a depleted GcMAF precursor in cancer
patients. Alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer
patient serum to activate macrophages.
Acknowledgments
Thanks to Dr. Douglas Lake of the School of Life Sciences
at Arizona State University for his generous donation of
pancreatic cancer patient plasma samples. Also, thanks
to Jim Tassano for helpful discussions.
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